Methods and compositions for isolating exosomes

Abstract

Disclosed are surprising new methods and compositions for isolating extracellular microvesicles such as exosomes, particularly disease-related and phosphatidylserine (PS)-positive extracellular microvesicles as exemplified by tumor- and viral-derived exosomes. The methods of the invention are rapid, efficient, cost-effective and, importantly, are suitable for use with large volumes of biological fluids and produce antigenically intact extracellular microvesicles and exosomes. The methods and compositions are based on the surprising use of acetate buffers to isolate large quantities of extracellular microvesicles, particularly tumor-derived exosomes, from solution, without damaging their morphological or functional properties or antigenicity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims priority to first U.S. provisional application Ser. No. 61 / 945,718, filed Feb. 27, 2014, and to second U.S. provisional application Ser. No. 61 / 970,529, filed Mar. 26, 2014, the entire disclosures of which applications, including the specification, claims and drawings are incorporated herein by reference without disclaimer.BACKGROUND OF THE INVENTION[0002]Field of the Invention[0003]The present invention relates to the fields of biotechnology, and particularly to extracellular microvesicles such as exosomes. The invention provides surprising new methods and compositions for isolating disease-related and phosphatidylserine (PS)-positive extracellular microvesicles such as tumor- and viral-derived exosomes, particularly tumor-derived exosomes, which are especially suitable for use with large volumes of biological fluids and produce extracellular microvesicles and exosomes that are antigenically intact.[0004]De...

AI Technical Summary

Benefits of technology

[0012]The present invention addresses the foregoing and other needs of the prior art by providing new methods and compositions for isolating extracellular microvesicles such as exosomes, particularly disease-related and phosphatidylserine (PS)-positive exosomes, including tumor-derived exosomes, which methods are rapid, efficient, cost-effective and suitable for use with large volumes of biological fluids. These methods are based on the surprising use of acetate buffers to isolate extracellular microvesicles such as exosomes from solution. The invention provides the ability to isolate large quantities of extracellular microvesicles and exosomes, particularly antigenically intact, disease-related and PS-positive exosomes such as tumor-derived exosomes, which are indistinguishable from those prepared by the current ultracentrifugation methods, the latter of which are time-consuming, cumbersome and volume-limited.

[0019]By applying the invention to all disease-related extracellular microvesicles, such as disease-related exosomes, phosphatidylserine will preferably be present on the surface of the extracellular microvesicles, more preferably in association with non-lipid membrane components, wherein the non-lipid membrane components comprise membrane proteins. In this regard, the acetate buffer is believed to neutralize the surface charge of the phosphatidylserine on the disease-related extracellular microvesicles, thereby precipitating the disease-related extracellular microvesicles from the biological fluid.

[0034]As the invention effectively separates disease-related extracellular microvesicles from biological fluids that contain mixed populations of extracellular microvesicles, the invention further provides methods to obtain disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant of diseased or tumor cells cultured in the presence of serum that contains normal extracellular microvesicles, preferably wherein the disease-related or tumor-derived extracellular microvesicles have negatively-charged phosphatidylserine on their surface. These methods comprise contacting the supernatant with an acetate buffer at a pH and concentration effective to selectively precipitate disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant and collecting the precipitate, thereby obtaining disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant without substantial contamination from normal extracellular microvesicles in the serum. A particular example is wherein mouse or human tumor cells are cultured in the presence of bovine or fetal bovine serum.

[0059]Acetate buffers comprising both sodium acetate and potassium acetate, and mixtures thereof, are effective. In certain embodiments, the acetate buffers are essentially free from volume excluding polymers, such as polyethylene glycol. An exemplary preferred embodiment is a sodium acetate buffer that has a pH of about 4.75 and a final concentration in the sample of about 0.1M.

Problems solved by technology

However, the available techniques are limited by drawbacks in two important respects.

Firstly, as applied to extracellular microvesicle and exosome preparation in general, they are time-consuming, cumbersome and / or costly, and limited by the amounts of material that can be processed.

The typical approach of concentrating the biological medium using ultracentrifugation before proceeding with exosome isolation is very time consuming and requires specialized laboratory equipment.

Secondly, the current techniques are particularly limited as they apply to tumor-derived extracellular microvesicle and exosomes.

In addition to the general drawbacks described above, such methods and kits are very limiting, requiring both advance knowledge of a particular exosome surface marker to be exploited in the antibody binding, as well as many detailed technical steps in the protocol, such as the preparation and use of biotinylated capture antibodies.

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